Helix-helix packing and interfacial pairwise interactions of residues in membrane proteins

Helix-helix packing plays a critical role in maintaining the tertiary structures of helical membrane proteins. By examining the overall distribution of voids and pockets in the transmembrane (TM) regions of helical membrane proteins, we found that bacteriorhodopsin and halorhodopsin are the most tightly packed, whereas mechanosensitive channel is the least tightly packed. Large residues F, W, and H have the highest propensity to be in a TM void or a pocket, whereas small residues such as S, G, A, and T are least likely to be found in a void or a pocket. The coordination number for non-bonded interactions for each of the residue types is found to correlate with the size of the residue. To assess specific interhelical interactions between residues, we have developed a new computational method to characterize nearest neighboring atoms that are in physical contact. Using an atom-based probabilistic model, we estimate the membrane helical interfacial pairwise (MHIP) propensity. We found that there are many residue pairs that have high propensity for interhelical interactions, but disulfide bonds are rarely found in the TM regions. The high propensity pairs include residue pairs between an aromatic residue and a basic residue (W-R, W-H, and Y-K). In addition, many residue pairs have high propensity to form interhelical polar-polar atomic contacts, for example, residue pairs between two ionizable residues, between one ionizable residue and one N or Q. Soluble proteins do not share this pattern of diverse polar-polar interhelical interaction. Exploratory analysis by clustering of the MHIP values suggests that residues similar in side-chain branchness, cyclic structures, and size tend to have correlated behavior in participating interhelical interactions. A chi-square test rejects the null hypothesis that membrane protein and soluble protein have the same distribution of interhelical pairwise propensity. This observation may help us to understand the folding mechanism of membrane proteins.     

Protein-protein interactions; coupling of structurally conserved residues and of hot spots across interfaces. Implications for docking

Hot spot residues contribute dominantly to protein-protein interactions. Statistically, conserved residues correlate with hot spots, and their occurrence can distinguish between binding sites and the remainder of the protein surface. The hot spot and conservation analyses have been carried out on one side of the interface. Here, we show that both experimental hot spots and conserved residues tend to couple across two-chain interfaces. Intriguingly, the local packing density around both hot spots and conserved residues is higher than expected. We further observe a correlation between local packing density and experimental deltadeltaG. Favorable conserved pairs include Gly coupled with aromatics, charged and polar residues, as well as aromatic residue coupling. Remarkably, charged residue couples are underrepresented. Overall, protein-protein interactions appear to consist of regions of high and low packing density, with the hot spots organized in the former. The high local packing density in binding interfaces is reminiscent of protein cores.     

High-resolution X-ray crystal structures of human gammaD crystallin (1.25 A) and the R58H mutant (1.15 A) associated with aculeiform cataract

Several human cataracts have been linked to mutations in the gamma crystallin gene. One of these is the aculeiform cataract, which is caused by an R58H mutation in gammaD crystallin. We have shown previously that this cataract is caused by crystallization of the mutant protein, which is an order of magnitude less soluble than the wild-type. Here, we report the very high-resolution crystal structures of the mutant and wild-type proteins. Both proteins crystallize in the same space group and lattice. Thus, a strict comparison of the protein-protein and protein-water intermolecular interactions in the two crystal lattices is possible. Overall, the differences between the mutant and wild-type structures are small. At position 58, the mutant protein loses the direct ion-pair intermolecular interaction present in the wild-type, due to the differences between histidine and arginine at the atomic level; the interaction in the mutant is mediated by water molecules. Away from the mutation site, the mutant and wild-type lattice structures differ in the identity of side-chains that occupy alternate conformations. Since the interactions in the crystal phase are very similar for the two proteins, we conclude that the reduction in the solubility of the mutant is mainly due to the effect of the R58H mutation in the solution phase. The results presented here are also important as they are the first high-resolution X-ray structures of human gamma crystallins.     

Ideal architecture of residue packing and its observation in protein structures.

A simple model of sphere packing has been investigated as an ideal model for long-range interactions for the packing of non-bonded residues in protein structures. By superposing all residues, the geometry of packing around a central residue is investigated. It is found that all residues conform almost perfectly to this lattice model for sphere packing when a radius of 6.5 A is used to define non-bonded (virtual) interacting residues. Side-chain positions with respect to sequential backbone segments are relatively regular as well. This lattice can readily be used in conformation simulations to reduce the conformational space.     

The key role of the residue 103 in the surface interactions of gamma-crystallins

A comparative study of intramolecular crystal interactions of two homologous gamma-crystallins II and IIIb from calf lens has been carried out. It has been shown that the key role in formation of "dimeric" associates of the head-to-tail type for gamma-crystallin IIIb is played by Met-103 which is located in the middle of the hydrophobic surface region. The absence of such a region in the molecule of gamma-crystallin II is explained by replacement of Met-103 by Ser-103. A similar alternative with the exchange of the hydrophobic residue by the hydrophilic one is observed for different gene products of gamma-crystallins from a number of vertebrates. This suggests intermolecular interaction of gamma-crystallins in the native medium of the lens.     

Mutational analysis of hydrophobic domain interactions in gamma B-crystallin from bovine eye lens.

gamma B-crystallin is a monomeric member of the beta gamma-superfamily of vertebrate eye lens proteins. It consists of two similar domains with all-beta Greek key topology associating about an approximate two-fold axis. At pH 2, with urea as the denaturant, the domains show independent equilibrium unfolding transitions, suggesting different intrinsic stabilities. Denaturation experiments using recombinant one- or two-domain proteins showed that the N-terminal domain on its own exhibits unaltered intrinsic stability but contributes significantly to the stability of its C-terminal partner. It has been suggested that docking of the domains is determined by a hydrophobic interface that includes phenylalanine at position 56 of the N-terminal domain. In order to test this hypothesis, F56 was substituted by site-directed mutagenesis in both complete gamma B-crystallin and its isolated N-terminal domain. All mutations destabilize the N-terminal domain to about the same extent but affect the C-terminal domain in a different way. Replacement by the small alanine side chain or the charged aspartic acid residue results in a significant destabilization of the C-terminal domain, whereas the more bulky tryptophan residue causes only a moderate decrease in stability. In the mutants F56A and F56D, equilibrium unfolding transitions obtained by circular dichroism and intrinsic fluorescence differ, suggesting a more complex denaturation behavior than the one observed for gamma B wild type. These results confirm how mutations in one crystallin domain can affect the stability of another when they occur at the interface. The results strongly suggest that size, hydrophobicity, and optimal packing of amino acids involved in these interactions are critical for the stability of gamma B-crystallin.     

Pitfalls in the interpretation of structural changes in mutant proteins from crystal structures

PpcA is a small protein with 71 residues that contains three covalently bound hemes. The structures of single mutants at residue 58 have shown larger deviations in another part of the protein molecule than at the site of the mutation. Closer examination of the crystal packing has revealed the origin of this unexpected structural change. The site of mutation is within Van der Waals distance from another protein molecule related by a crystallographic twofold axis within the crystal. The structural changes occurred at or near the mutation site have led to a slight adjustment of the surface residues in contact. The observed deviations between the native and the mutant molecular structures are derived from the new crystal packing even though the two crystals are essentially isomorphous. Without careful consideration of the crystal lattice a non-expert looking at only the coordinates deposited in the Protein Data Bank could draw erroneous conclusion that mutation in one part of the molecule affected the structure of the protein in a distant part of the molecule.     

High resolution structure of an oligomeric eye lens beta-crystallin. Loops, arches, linkers and interfaces in beta B2 dimer compared to a monomeric gamma-crystallin.

beta-Crystallins are polydisperse, oligomeric structural proteins that have a major role in forming the high refractive index of the eye lens. Using single crystal X-ray crystallography with molecular replacement, the structure of beta B2 dimer has been solved at 2.1 A resolution. Each subunit comprises an N and C-terminal domain that are very similar and each domain is formed from two similar "Greek key" motifs related by a local dyad. Sequence differences in the internally quadruplicated molecules, analysed in terms of their beta-sheets, hairpins and arches, give rise to structural differences in the motifs. Whereas the related family of gamma-crystallins are monomers, beta-crystallins are always oligomers. In the beta B2 subunit, the domains, each comprising two motifs, are separated by an extended linking peptide. A crystallographic 2-fold axis relates the two subunits of the dimer so that the N-terminal domain of one subunit of beta B2 and the C-terminal domain of the symmetry-related subunit are topologically equivalent to the two covalently connected domains of gamma B-crystallin. The intersubunit domain interface is very similar to the intradomain interface of gamma B, although many sequence differences have resulted in an increase in polar interactions between domains in beta B2. Comparison of the structures of beta B2 and gamma B-crystallins shows that the two families differ largely in the conformation of their connecting peptides. A further extensive lattice contact indicates a tetramer with 222 symmetry. The ways in which insertions and extensions in the beta-crystallin effect oligomer interactions are described. The two kinds of crystallin are analysed for structural features that account for their different stabilities. These studies are a basis for understanding formation of higher aggregates in the lens.     

Rat lens gamma-crystallins. Characterization of the six gene products and their spatial and temporal distribution resulting from differential synthesis

We have isolated, purified and characterized six individual gamma-crystallin polypeptides present in the rat lens. Comparison of their amino acid compositions with the known structure of the six gamma-crystallin genes permits a one-to-one correspondence to be made between each protein synthesized and the encoding gene. This demonstrates that each of the six genes is actually expressed in vivo. Two classes of three gamma-crystallins each, which we have designated classes gamma ABC and gamma DEF, are known to exist, on the basis of internal sequence homology. We have measured the temperature-dependent phase-separation characteristics of solutions of the six purified gamma-crystallins, and find that the three members of the gamma DEF class (gamma 2-2, gamma 3-1 and gamma 4-1) are all cryo-proteins with relatively high phase-separation temperatures, whereas the three gamma ABC crystallins (gamma 1-1, gamma 1-2 and gamma 2-1) do not show phase separation above -7 degrees C. We have measured the spatial distribution in rat lens of each of the alpha-, beta- and gamma-crystallins as a function of age from 1 to 420 days, using size-exclusion and ion-exchange high-pressure liquid chromatography (HPLC). Our findings in the cortical layer permit us to establish the differential synthesis of each of the crystallins during lens development. Particular attention has been devoted to the spatial and temporal distribution of the six individual gamma-crystallins. Up to birth, synthesis of the three components of the gamma DEF class predominates, and in particular that of gamma 2-2. In subsequent development the three components of the gamma ABC class assume a greater proportion of monomeric crystallins synthesized, while beta s-crystallin synthesis predominates in late development. Our analysis of different layers within single lenses provides novel information on spatial gradients of the water-soluble and water-insoluble protein fractions as a function of age. We consider the consequences of these findings for lens transparency and opacity in both rat and mouse lens. We show that the high concentrations of gamma DEF-crystallins appear to be responsible for the opacity known to occur in young rat lenses. We conclude from these observations that close control of the differential synthesis of gamma-crystallins plays an important role in maintaining lens transparency during development.     

The role of packing interactions in stabilizing folded proteins.

In order to investigate the role of nonpolar side chains in determining protein stability, we have carried out a molecular dynamics simulation study of the thermodynamics of interconverting isoleucine and valine side chains in the core of ribonuclease T1. The free energy change in the unfolded state, which we take to be fully solvated, was small and agrees qualitatively with experimental studies of alkane solvation. In the two Ile----Val mutations studied, the protein was able to relax around the smaller side chains, while in the case of the two Val----Ile mutations, the ability of the core to accommodate the extra methylene group depended on where the mutation took place. We argue that the experimentally observed decrease in stability for mutating isoleucine into valine results from a loss of favorable packing interactions of the side chain in the folded form of the protein. This supports the view that packing interactions in the folded state are an important contributor to the overall stability of the folded protein and that the core of the native protein is packed efficiently and almost completely.     

Role of medium--and long-range interactions in discriminating globular and membrane proteins.

The analysis of inter-residue interactions in protein structures provides considerable insight to understand their folding and stability. We have previously analyzed the role of medium- and long-range interactions in the folding of globular proteins. In this work, we study the distinct role of such interactions in the three-dimensional structures of membrane proteins. We observed a higher number of long-range contacts in the termini of transmembrane helical (TMH) segments, implying their role in the stabilization of helix-helix interactions. The transmembrane strand (TMS) proteins are having appreciably higher long-range contacts than that in all-beta class of globular proteins, indicating closer packing of the strands in TMS proteins. The residues in membrane spanning segments of TMH proteins have 1.3 times higher medium-range contacts than long-range contacts whereas that of TMS proteins have 14 times higher long-range contacts than medium-range contacts. Residue-wise analysis indicates that in TMH proteins, the residues Cys, Glu, Gly, Pro, Gln, Ser and Tyr have higher long-range contacts than medium-range contacts in contrast with all-alpha class of globular proteins. The charged residue pairs have higher medium-range contacts in all-alpha proteins, whereas hydrophobic residue pairs are dominant in TMH proteins. The information on the preference of residue pairs to form medium-range contacts has been successfully used to discriminate the TMH proteins from all-alpha proteins. The statistical significance of the results obtained from the present study has been verified using randomized structures of TMH and TMS protein templates.     

Role of helix-helix interactions in assembly of the bacteriorhodopsin lattice.

The purple membrane of Halobacterium salinarium is a two-dimensional lattice of lipids and the integral membrane protein bacteriorhodopsin (BR). To determine whether helix-helix interactions within the membrane core stabilize this complex, we substituted amino acid residues at the helix-helix interface between BR monomers and examined the assembly of the protein into the lattice. Lattice assembly was demonstrated to fit a cooperative self-assembly model that exhibits a critical concentration in vivo. Using this model as the basis for a quantitative assay of lattice stability, bulky substitutions at the helix-helix interface between BR monomers within the membrane core were shown to be destabilizing, probably due to steric clash. Ala substitutions of two residues at the helix-helix interface also reduced stability, suggesting that the side chains of these residues participate in favorable van der Waals packing interactions. However, the stabilizing interactions were restricted to a small region of the interface, and most of the substitutions had little effect. Thus, the contribution of helix-helix interactions to lattice stability appears limited, and favorable interactions between other regions of neighboring BR monomers or between BR and lipid molecules must also contribute.     

Energetics of charge-charge interactions between residues adjacent in sequence

Statistical analysis of the residue separation between a pair of ionizable side chains within 4 ? of each other was performed on a set of 1560 non-homologous PDB structures. We found that the frequency of pairs of like charges (i.e., pairs consisting of acidic residues Asp and Glu or pairs consisting of basic residues Arg and Lys) is two orders of magnitude lower than the pairs of oppositely charged residues (salt-bridges). We also found that for pairs of like charges the distribution is skewed dramatically towards short residue separation (<3). On the basis of these observations, we hypothesize that at short residue separation the repulsion between charges does not contribute much to the protein stability and the effects are largely dominated by the long range charge-charge interactions with other ionizable groups in the protein molecule. To test this hypothesis, we incorporated various pairs of charged residues at position 63 and 64 of ubiquitin and compared the stabilities of these variants. We also performed calculations of the expected changes in the charge-charge interactions. A very good correlation between experimental changes in the stability of ubiquitin variants, and changes in the energy of charge-charge interactions provides support for the hypothesis that a pair of ionizable residues next to each other in sequence modulates protein stability via long range charge-charge interactions with the rest of the protein.     

The effect of long-range interactions on the secondary structure formation of proteins

The influence of long-range residue interactions on defining secondary structure in a protein has long been discussed and is often cited as the current limitation to accurate secondary structure prediction. There are several experimental examples where a local sequence alone is not sufficient to determine its secondary structure, but a comprehensive survey on a large data set has not yet been done. Interestingly, some earlier studies denied the negative effect of long-range interactions on secondary structure prediction accuracy. Here, we have introduced the residue contact order (RCO), which directly indicates the separation of contacting residues in terms of the position in the sequence, and examined the relationship between the RCO and the prediction accuracy. A large data set of 2777 nonhomologous proteins was used in our analysis. Unlike previous studies, we do find that prediction accuracy drops as residues have contacts with more distant residues. Moreover, this negative correlation between the RCO and the prediction accuracy was found not only for beta-strands, but also for alpha-helices. The prediction accuracy of beta-strands is lower if residues have a high RCO or a low RCO, which corresponds to the situation that a beta-sheet is formed by beta-strands from different chains in a protein complex. The reason why the current study draws the opposite conclusion from the previous studies is examined. The implication for protein folding is also discussed.     

Role of the B helix in early folding events in apomyoglobin: evidence from site-directed mutagenesis for native-like long range interactions

The folding pathways of four mutants in which bulky hydrophobic residues in the B helix of apomyoglobin (ApoMb) are replaced by alanine (I28A, L29A, I30A, and L32A) have been analyzed using equilibrium and kinetic methods employing NMR, CD, fluorescence and mass spectrometry. Hydrogen exchange pulse-labeling followed by mass spectrometry reveals detectable intermediates in the kinetic folding pathways of each of these mutants. Comparison of the quench-flow data analyzed by NMR for the wild-type protein and the mutants showed that the substitutions I28A, L29A and L32A lead to destabilization of the B helix in the burst phase kinetic intermediate, relative to wild-type apomyoglobin. In contrast, the I30A mutation apparently has a slight stabilizing effect on the B helix in the burst phase intermediate; under weak labeling conditions, residues in the C helix region were also relatively stabilized in the mutant compared to the wild-type protein. This suggests that native-like helix B/helix C packing interactions occur in the folding intermediate. The L32A mutant showed significantly lower proton occupancies in the burst phase for several residues in the G helix, specifically F106, I107, E109 and A110, which are in close proximity to L32 in the X-ray structure of myoglobin, providing direct evidence that native-like helix B/helix G contacts are formed in the apomyoglobin burst phase intermediate. The L29A mutation resulted in an increase in burst phase proton occupancies for several residues in the E helix. Since these regions of the B and E helices are not in contact in the native myoglobin structure, these effects suggest the possibility of non-native B/E packing interactions in the kinetic intermediate. The differing effects of these B helix mutations on the apomyoglobin folding process suggests that each side-chain plays a different and important role in forming stable structure in the burst phase intermediate, and points to a role for both native-like and non-native contacts in stabilization of the folding intermediate.     

Lipid-cholesterol interactions in the P beta' phase. Application of a statistical mechanical model.

We describe a statistical mechanical model for lipid-cholesterol mixtures in the P beta' (ripple) phase of lipid bilayers. The model is a simple extension of an earlier model for the ripple phase in pure lipid bilayers. The extension consists of adding a degree of freedom to allow for the occupation of underlying lattice sites by cholesterol molecules, and adding a lipid-cholesterol interaction term to the model Hamiltonian. The interaction term was constructed based on numerical calculations of lipid-cholesterol energies for several different packing juxtapositions of the two molecules. Other than the lipid-cholesterol interactions, the extended model uses the same parameter set as the earlier model, so that comparison of the properties of the extended model with experimental data serves as a test of the validity of the original model. Properties of the model were calculated using the Monte Carlo method. Results are displayed as snapshots of the ripple configurations at different cholesterol concentrations. The spacing of the ripples increases with increasing cholesterol concentration and the rate of increase compares very well with experimental data. The success of this model supports the conclusion drawn earlier that frustration arising from anisotropic packing interactions is responsible for the ripple phase in lipid bilayers. In the extended model these packing interactions are responsible for the selective partitioning of cholesterol in the regions between the ripples.     

Surface interactions of gamma-crystallins in the crystal medium in relation to their association in the eye lens

A comparative study of intermolecular interactions in crystals of two homologous low molecular weight proteins, gamma-II and gamma-IIIb crystallins, from calf eye lens was carried out. Crystal packings for these proteins are very different: intermolecular contact areas compose about 33% of the total accessible surface area of gamma-II as compared with 13% in gamma-III. Two key residues seem to be mainly responsible for the differences in protein association in the crystal medium. These are Ser 103 and Leu 155 in gamma-II, which are replaced by Met 103 and His 155 in gamma-IIb. A similar substitution of these residues is observed in different gene products of gamma-crystallins from a number of vertebrates. This is consistent with the existence of a genetically controlled mechanism for determining intermolecular association of gamma-crystallins in the native medium of the lens.     

Crystal structure of thrombin in a self-inhibited conformation

The activating effect of Na(+) on thrombin is allosteric and depends on the conformational transition from a low activity Na(+)-free (slow) form to a high activity Na(+)-bound (fast) form. The structures of these active forms have been solved. Recent structures of thrombin obtained in the absence of Na(+) have also documented inactive conformations that presumably exist in equilibrium with the active slow form. The validity of these inactive slow form structures, however, is called into question by the presence of packing interactions involving the Na(+) site and the active site regions. Here, we report a 1.87A resolution structure of thrombin in the absence of inhibitors and salts with a single molecule in the asymmetric unit and devoid of significant packing interactions in regions involved in the allosteric slow --> fast transition. The structure shows an unprecedented self-inhibited conformation where Trp-215 and Arg-221a relocate >10A to occlude the active site and the primary specificity pocket, and the guanidinium group of Arg-187 penetrates the protein core to fill the empty Na(+)-binding site. The extreme mobility of Trp-215 was investigated further with the W215P mutation. Remarkably, the mutation significantly compromises cleavage of the anticoagulant protein C but has no effect on the hydrolysis of fibrinogen and PAR1. These findings demonstrate that thrombin may assume an inactive conformation in the absence of Na(+) and that its procoagulant and anticoagulant activities are closely linked to the mobility of residue 215.